Fig. 4: Patient 1-derived BLOC1S5 transgenes do not restore pigmentation to mouse Bloc1s5mu/mu melanocytes.
From: BLOC1S5 pathogenic variants cause a new type of Hermansky–Pudlak syndrome
(a–h) Melanin content of fixed mouse melanocyte cell lines melan-Ink4a (wild-type), melan-mu (from Bloc1s5mu/mu mice) or melan-mu stably expressing the indicated HA.11-tagged Muted (BLOC1S5) or Pallidin (BLOC1S6) transgenes was assessed by bright field microscopy. Unpigmented cells are outlined in white. Scale: 10 µm. (i) Melanin content in lysates from the indicated cell lines was assayed by spectrophotometry. Plat-E is a nonpigmented cell line used as a negative control. Data are normalized to values from melan-mu and represent mean ± SEM from at least three experiments. Statistical analysis was performed using the Kruskal–Wallis test by ranks. *P < 0.05; ****P < 0.0001. (j) Reverse-transcription polymerase chain reaction (RT-PCR) analysis to confirm transgene expression. Complementary DNA (cDNA) was amplified from each of the indicated cell lines using primers for the hygromycin resistance gene expressed from the internal ribosome entry site of the pBMN-Hygro-IRES retroviral backbone, and products were fractionated by agarose gel electrophoresis. Shown is a representative of two experiments. Positions of DNA markers (bp) are shown at left. (k) Whole-cell lysates of indicated cell lines fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were immunoblotted for the HA.11 epitope tag, BLOC-1 subunits Dysbindin and Pallidin, or γ-tubulin as a control. Shown is a representative of three experiments. Relevant bands (right) and positions of molecular weight markers (kDa, left) are indicated. Note that the higher molecular weight band in the Pallidin blot is HA-tagged Pallidin, whereas the lower molecular weight band represents endogenous Pallidin.
